Novel chemokine-like polypeptides

ABSTRACT

The present invention discloses open reading frames (ORFs) in human genome encoding for novel chemokines-like polypeptides, and reagents related thereto including variants, mutants and fragments of said polypeptides, as well as ligands and antagonists directed against them. The invention provides methods for identifying and making these molecules, for preparing pharmaceutical compositions containing them, and for using them in the diagnosis, prevention and treatment of diseases.

FIELD OF THE INVENTION

The present invention relates to nucleic acid sequences identified inhuman genome as encoding for novel polypeptides, more specifically forchemokine-like polypeptides.

BACKGROUND OF THE INVENTION

The mammalian immune response is based on a series of complex,network-like interactions involving cellular components (such aslymphocytes or granulocytes) and soluble proteins, capable of modulatingcellular activities (movement, proliferation, differentiation, etc.).Thus, there is considerable interest in the isolation andcharacterization of cell modulating factors, with the purpose ofproviding significant advancements in the diagnosis, prevention, andtherapy of human disorders, in particular the ones associated to theimmune system.

Chemokines are amongst these soluble proteins, since they are involvedin the directional migration and activation of cells. This superfamilyof small (70-130 amino acids), secreted, heparin-binding,pro-inflammatory proteins is known especially for the role in theextravasation of leukocytes from the blood to tissue localizationsneeding the recruitment of these cells (Baggiolini M et al., 1997;Yoshie O F et al., 2001; Fernandez E J and Lolis E, 2002).

Chemokines are not only functionally related but also structurallyrelated, since they all contain a central region in which conservedCysteines form intramolecular bonds. In particular, the number and theposition of the most N-terminal of these conserved Cysteines in themature polypeptides is the basic criteria for the generally recognizedclassification of chemokines, essentially divided between chemokineshaving a single or adjacent Cysteines (C—C chemokines), or chemokineshaving two Cysteines separated by 1-3 amino acids (C—X—C chemokines).

A series of membrane receptors, all heptahelical G-protein coupledreceptors, are the binding partners that allow chemokines to exert theirbiological activity on the target cells. The physiological effects ofchemokines result from a complex and integrated system of concurrentinteractions. Different cells can present specific combinations ofreceptors according to their state and/or type. Moreover, chemokinereceptors often have overlapping ligand specificity, so that a singlereceptor can bind different chemokines, as well a single chemokine canbind different receptors, still at high affinity.

Usually chemokines are produced at the site of an injury, inflammation,or other tissue alteration, and exert their activity in a paracrine orautocrine fashion. However, cell-type specific migration and activationin inflammatory and immune processes is not the sole activity ofchemokines. Other physiological activities, such as hematopoiesis orangiogenesis, and pathological conditions, such as metastasis,transplant rejection, Alzehimer's disease or atherosclerosis, appear tobe regulated by, at least, some of these proteins. In fact, chemokinesand/or their receptors have been found considerably over-expressedand/or activated in several animal models or clinical samples (Haskell CA et al., 2002; Lucas A D and Greaves D R, 2001; Frederick M J andClayman G L, 2001; Godessart N and Kunkel S L, 2001; Reape T J and GrootP H, 1999).

There are potential drawbacks in using chemokines as therapeutic agents(tendency to aggregate and promiscuous binding, in particular), butmolecules having antagonistic properties against chemokines are widelyconsidered as offering valuable opportunities for therapeuticintervention in disorders associated to excessive chemokine activities.The inhibition of specific chemokines and their receptors is considereda solution for preventing undesirable or uncontrolled cellularprocesses, such as recruitment or activation (Baggiolini M, 2001;Proudfoot A, 2000; Rossi D F and Zlotnik A, 2000).

The extensive sequencing programs and bioinformatics have made availablea large amount of tools and information on human genome and physiology(Quinn-Senger K E et al., 2002; Browne M J, 2000). Such technologieswere also used for discovering novel chemokines and receptors possiblyproviding new and useful therapeutic molecules and targets. Initially,chemokines genes were regularly mapped on chromosomes 4 and 17, ingene-rich areas of human genome (Nomiyama H et al., 2001), but theliterature provides various approaches for characterizing novelchemokines by comparing the tissue-distribution of transcripts.Chemokines are usually expressed in lymphoid and other tissues but novelchemokines can have specific expression patterns and can be mapped tochromosomal loci different from the traditional gene clusters (WO02/70706; Wells T N and Peitsch M C, 2000; Chantry D F et al., 1998;Rossi D et al., 1997).

Novel chemokines have been identified by applying strict homologycriteria to known chemokines. However, since the actual content inpolypeptide-encoding sequence in human genome for chemokines (as for anyother protein family) is still unknown, the possibility still exists toidentify DNA sequence encoding for polypeptides having chemotacticactivities by applying alternative criteria in the analysis of OpenReading Frames (ORFs, that is, DNA sequences containing consecutivecoding triplets of nucleotides, not interrupted by a termination codonand that can be potentially translated in a polypeptide) present inhuman genome.

SUMMARY OF THE INVENTION

The invention is based upon the identification of Open Reading Frames(ORFs) in human genome encoding novel chemokine-like polypeptides.

Accordingly, the invention provides isolated polypeptides having theamino acid sequence given by SEQ ID NO: 2, 4, 6, 8, 10, 12, 14 or 16,and their mature forms, variants, and fragments, as polypeptides havingchemotactic activity. The invention includes also the nucleic acidsencoding them, vectors containing such nucleic acids, and cellcontaining these vectors or nucleic acids, as well as other relatedreagents such as fusion proteins, ligands, and antagonists.

The invention provides methods for identifying and making thesemolecules, for preparing pharmaceutical compositions containing them,and for using them in the diagnosis, prevention and treatment ofdiseases.

DESCRIPTION OF THE FIGURES

FIG. 1: alignment of the ORF contained in the DNA sequence GNSQ_(—)1754(SEQ ID NO: 1) with the protein sequence p1754 (SEQ ID NO: 2). Thepredicted N-terminal signal sequence is boxed. The predicted C-terminalalpha helix is underlined. The codons matching the original selectioncriteria are indicated with §. The arrows indicate the position of theprimers CL_(—)1754_(—)5 (forward) and CL_(—)1754_(—)3 (reverse) in theORF sequence.

FIG. 2: alignment of the ORF contained in the DNA sequence GNSQ_(—)0711(SEQ ID NO: 3) with the protein sequence p0711 (SEQ ID NO: 4). Thepredicted N-terminal signal sequence is boxed. The predicted C-terminalalpha helix is underlined. The codons matching the original selectioncriteria are indicated with §. The arrows indicate the position of theprimers CL_(—)0711_(—)5 (forward) and CL_(—)0711_(—)3 (reverse) in theORF sequence.

FIG. 3: alignment of the ORF contained in the DNA sequence GNSQ_(—)2882(SEQ ID NO: 5) with the protein sequence p2882 (SEQ ID NO: 6). Thepredicted N-terminal signal sequence is boxed. The predicted C-terminalalpha helix is underlined. The codons matching the original selectioncriteria are indicated with §.

FIG. 4: alignment of the ORF contained in the DNA sequence GNSQ_(—)4711(SEQ ID NO: 7) with the protein sequence p4711 (SEQ ID NO: 8). Thepredicted N-terminal signal sequence is boxed. The predicted C-terminalalpha helix is underlined. The codons matching the original selectioncriteria are indicated with §.

FIG. 5: alignment of the ORF contained in the DNA sequence GNSQ_(—)4320(SEQ ID NO: 9) with the protein sequence p4320 (SEQ ID NO: 10). Thepredicted N-terminal signal sequence is boxed. The predicted C-terminalalpha helix is underlined. The codons matching the original selectioncriteria are indicated with §. The arrows indicate the position of theprimers CL_(—)4320_(—)5 (forward) and CL_(—)4320_(—)3 (reverse) in theORF sequence.

FIG. 6: alignment of the ORF contained in the DNA sequence GNSQ_(—)5008(SEQ ID NO: 11) with the protein sequence p5008 (SEQ ID NO: 12). Thepredicted N-terminal signal sequence is boxed. The predicted C-terminalalpha helix is underlined. The codons matching the original selectioncriteria are indicated with §. The arrows indicate the position of theprimers CL_(—)5008_(—)5 (forward) and CL_(—)5008_(—)3 (reverse) in theORF sequence.

FIG. 7: alignment of the ORF contained in the DNA sequence GNSQ_(—)0210(SEQ ID NO: 13) with the protein sequence p0210 (SEQ ID NO: 14). Thepredicted N-terminal signal sequence is boxed. The predicted C-terminalalpha helix is underlined. The codons matching the original selectioncriteria are indicated with §. The arrows indicate the position of theprimers CL_(—)0210_(—)5 (forward) and CL_(—)0210_(—)3 (reverse) in theORF sequence.

FIG. 8: alignment of the ORF contained in the DNA sequence GNSQ_(—)4922(SEQ ID NO: 15) with the protein sequence p4922 (SEQ ID NO: 16). Thepredicted N-terminal signal sequence is boxed. The predicted C-terminalalpha helix is underlined. The codons matching the original selectioncriteria are indicated with §. The arrows indicate the position of theprimers CL_(—)4922_(—)5 (forward) and CL_(—)4922_(—)3 (reverse) in theORF sequence.

FIG. 9: alignment of human CXCL chemokines with the CXC chemokine-likeprotein sequences of the invention p1754 (SEQ ID NO: 2), p0711 (SEQ IDNO: 4), p2882 (SEQ ID NO: 6), p0210 (SEQ ID NO: 14), and p4922 (SEQ IDNO: 16). The following human CXCL chemokines have been considered: CXCL1(SWISSPROT Acc. No P09341), CXCL2 (SWISSPROT Acc. No P19875), CXCL3(SWISSPROT Acc. No NP_(—)002081), CXCL4 (SWISSPROT Acc. NoNP_(—)002610), CXCL5 (SWISSPROT Acc. No P42830), CXCL6 (SWISSPROT Acc.N° P80162), CXCL7 (SWISSPROT Acc. No P02775), CXCL8 (SWISSPROT Acc. NoP10145), CXCL9 (SWISSPROT Acc. N° Q07325), CXCL10 (SWISSPROT Acc. N°P02778), CXCL11 (SWISSPROT Acc. No O14625). The protein sequences aredivided according to the structure of the three main regions: theN-terminal region (containing the signal sequence), the central Cys-richregion (containing the conserved Cysteines matching the originalselection criteria and indicated with §), and the C-terminal region(containing the predicted alpha helix).

FIG. 10: alignment of human CCL chemokines with the CXC chemokine-likeprotein sequences of the invention p4711 (SEQ ID NO: 8), p4320 (SEQ IDNO: 10), and GNSQ_(—)5008 (SEQ ID NO: 12). The following human CCLchemokines have been considered: CCL1 (SWISSPROT Acc. No P22362), CCL2(SWISSPROT Acc. No P13500), CCL3 (SWISSPROT Acc. No P10147), CCL4(SWISSPROT Acc. No P13236), CCL5 (SWISSPROT Acc. No P13501), CCL7(SWISSPROT Acc. No P80098), CCL8 (SWISSPROT Acc. No P80075). The proteinsequences are divided according to the structure of the three mainregions: the N-terminal region (containing the signal sequence), thecentral Cys-rich region (containing the conserved Cysteines matching theoriginal selection criteria and indicated with §), and the C-terminalregion (containing the predicted alpha helix).

FIG. 11: Map of the pEAK12d expression vector.

DETAILED DESCRIPTION OF THE INVENTION

The main object of the present invention is to provide novel, isolatedpolypeptides having chemotactic activity selected from the groupconsisting of:

-   -   a) the amino acid sequences SEQ ID NO: 2, 4, 6, 8, 10, 12, 14 or        16;    -   b) the mature form of the polypeptides SEQ ID NO: 2, 4, 6, 8,        10, 12, 14 or 16;    -   c) the polypeptides comprising the Cysteine-rich region of SEQ        ID NO: 2, 4, 6, 8, 10, 12, 14 or 16, as indicated in FIGS. 9 and        10;    -   d) the active variant of the amino acid sequence given by SEQ ID        NO: 2, 4, 6, 8, 10, 12, 14 or 16 wherein any amino acid        specified in the chosen sequence is non-conservatively        substituted, provided that no more than 15% of the amino acid        residues in the sequence are so changed;    -   e) the active fragments, precursors, salts, or derivatives of        the amino acid sequences given in a) to d).

The novel polypeptides p1754 (SEQ ID NO: 2), p0711 (SEQ ID NO: 4), p2882(SEQ ID NO: 6), p4711 (SEQ ID NO: 8), p4320 (SEQ ID NO: 10), p5008 ( SEQID NO: 12), p0210 (SEQ ID NO: 14), and p4922 (SEQ ID NO: 16) wereidentified on the basis of a consensus sequence for human chemokines inwhich the number and the position of selected amino acids (initialmethionine, cysteines, and hydrophobic residues) are defined for proteinsequence having length comparable to known chemokines.

The totality of amino acid sequences obtained by translating the knownORFs in the human genome were challenged using this consensus sequence,and the positive hits were further screened for the presence ofpredicted specific structural and functional “signatures” (a N-terminalsignal sequence and a C-terminal alpha helix), and finally selected bycomparing sequence features with known chemokines. Therefore, the novelpolypeptides of the invention can be predicted to have chemotacticactivities.

The terms “active” and “activity” refer to the chemotactic-likeproperties predicted for the chemokine-like amino acid sequences SEQ IDNO: 2, 4, 6, 8, 10, 12 14, or 16 in the present patent application.

Protein sequences having the indicated number of non-conservativesubstitutions can be identified using commonly available bioinformatictools (Mulder N J and Apweiler R, 2002; Rehm B H, 2001).

In addition to such sequences, a series of polypeptides forms part ofthe disclosure of the invention. Being chemokines known to go throughmaturation processes including the proteolytic removal of N-terminalsequences (by signal peptidases and other proteolytic enzymes), thepresent patent application also claim the mature form of thepolypeptides SEQ ID NO: 2, 4, 6, 8, 10, 12, 14 or 16. As mature form isintended any polypeptide showing chemotactic activity and resulting fromin vivo (by the expressing cells or animals) or in vitro (by modifyingthe purified polypeptides with specific enzymes) post-translationalmaturation processes. Mature forms of chemokines resulting fromC-terminal processing are also known (Ehlert J E et al., 1998). Otheralternative mature forms can also result from the addition of chemicalgroups such as sugars or phosphates.

A further group of polypeptides of the invention are the polypeptidescomprising the Cysteine-rich region of SEQ ID NO: 2, 4, 6, 8, 10, 12, 14or 16, as indicated in FIG. 9 and 10, since the central Cysteine-richregion contains the essential structural and functional groups ofchemokines.

Other claimed polypeptides are the active variants of the amino acidsequences given by SEQ ID NO: 2, 4, 6, 8, 10, 12 14, or 16 wherein anyamino acid specified in the chosen sequence is non-conservativelysubstituted, provided that no more than 15% of the amino acid residuesin the sequence are so changed. The indicated percentage has to bemeasured over the novel amino acid sequences disclosed in FIGS. 1-8, andin particular over a segment of at least 40 amino acids containing theCysteine-rich regions as indicated in FIGS. 9 and 10.

In accordance with the present invention, any substitution should bepreferably a “conservative” or “safe” substitution, which is commonlydefined a substitution introducing an amino acids having sufficientlysimilar chemical properties (e.g. a basic, positively charged amino acidshould be replaced by another basic, positively charged amino acid), inorder to preserve the structure and the biological function of themolecule.

The literature provide many models on which the selection ofconservative amino acids substitutions can be performed on the basis ofstatistical and physico-chemical studies on the sequence and/or thestructure of proteins (Rogov S I and Nekrasov A N, 2001). Protein designexperiments have shown that the use of specific subsets of amino acidscan produce foldable and active proteins, helping in the classificationof amino acid “synonymous” substitutions which can be more easilyaccommodated in protein structure, and which can be used to detectfunctional and structural homologs and paralogs (Murphy L R et al.,2000). The groups of synonymous amino acids and the groups of morepreferred synonymous amino acids are shown in Table I.

Active variants having comparable, or even improved, activity withrespect of corresponding chemokines may result from conventionalmutagenesis technique of the encoding DNA, from combinatorialtechnologies at the level of encoding DNA sequence (such as DNAshuffling, phage display/selection), or from computer-aided designstudies, followed by the validation for the desired activities asdescribed in the prior art.

Specific, non-conservative mutations can be also introduced in thepolypeptides of the invention with different purposes. Mutationsreducing the affinity of the chemokine-like polypeptide for a receptormay increase its ability to be reused and recycled, potentiallyincreasing its therapeutic potency (Robinson C R, 2002). Immunogenicepitopes eventually present in the polypeptides of the invention can beexploited for developing vaccines (Stevanovic S, 2002), or eliminated bymodifying their sequence following known methods for selecting mutationsfor increasing protein stability, and correcting them (van den Burg Band Eijsink V, 2002; WO 02/05146, WO 00/34317, WO 98/52976).

Further alternative polypeptides of the invention are active fragments,precursors, salt, or derivative of the amino acid sequences the abovedescribed sequences.

Fragments should present deletions of terminal or internal amino acidsnot altering their function, and should involve generally a few aminoacids, e.g., under ten, and preferably under three, without removing ordisplacing amino acids which are critical to the functional conformationof the proteins.

The “precursors” are compounds which can be converted into the compoundsof present invention by metabolic and enzymatic processing prior orafter the administration to the cells or to the body.

The term “salts” herein refers to both salts of carboxyl groups and toacid addition salts of amino groups of the polypeptides of the presentinvention. Salts of a carboxyl group may be formed by means known in theart and include inorganic salts, for example, sodium, calcium, ammonium,ferric or zinc salts, and the like, and salts with organic bases asthose formed, for example, with amines, such as triethanolamine,arginine or lysine, piperidine, procaine and the like. Acid additionsalts include, for example, salts with mineral acids such as, forexample, hydrochloric acid or sulfuric acid, and salts with organicacids such as, for example, acetic acid or oxalic acid. Any of suchsalts should have substantially similar activity to the peptides andpolypeptides of the invention or their analogs.

The term “derivatives” as herein used refers to derivatives which can beprepared from the functional groups present on the lateral chains of theamino acid moieties or on the amino- or carboxy-terminal groupsaccording to known methods. Such molecules can result also from othermodifications which do not normally alter primary sequence, for examplein vivo or in vitro chemical derivativization of polypeptides(acetylation or carboxylation), those made by modifying the pattern ofphosphorylation (introduction of phosphotyrosine, phosphoserine, orphosphothreonine residues) or glycosylation (by exposing the polypeptideto mammalian glycosylating enzymes) of a peptide during its synthesisand processing or in further processing steps. Alternatively,derivatives may include esters or aliphatic amides of thecarboxyl-groups and N-acyl derivatives of free amino groups or O-acylderivatives of free hydroxyl-groups and are formed with acyl-groups asfor example alcanoyl- or aryl-groups.

The generation of the derivatives may involve a site-directedmodification of an appropriate residue, in an internal or terminalposition. The residues used for attachment should they have a side-chainamenable for polymer attachment (i.e., the side chain of an amino acidbearing a functional group, e.g. lysine, aspartic acid, glutamic acid,cysteine, histidine, etc.). Alternatively, a residue having a side chainamenable for polymer attachment can replace an amino acid of thepolypeptide, or can be added in an internal or terminal position of thepolypeptide. Also, the side chains of the genetically encoded aminoacids can be chemically modified for polymer attachment, or unnaturalamino acids with appropriate side chain functional groups can beemployed. The preferred method of attachment employs a combination ofpeptide synthesis and chemical ligation. Advantageously, the attachmentof a water-soluble polymer will be through a biodegradable linker,especially at the amino-terminal region of a protein. Such modificationacts to provide the protein in a precursor (or “pro-drug”) form, that,upon degradation of the linker releases the protein without polymermodification.

Polymer attachment may be not only to the side chain of the amino acidnaturally occurring in a specific position of the antagonist or to theside chain of a natural or unnatural amino acid that replaces the aminoacid naturally occurring in a specific position of the antagonist, butalso to a carbohydrate or other moiety that is attached to the sidechain of the amino acid at the target position. Rare or unnatural aminoacids can be also introduced by expressing the protein in specificallyengineered bacterial strains (Bock A, 2001).

Variants of the polypeptides above indicated can be naturally occurring,being identified in organisms other than humans, or resulting from thetranslation of a single nucleotide polymorphism. Alternatively,artificial variants can be prepared by chemical synthesis, bysite-directed mutagenesis techniques, or any other known techniquesuitable thereof, which provide a finite set of substantiallycorresponding mutated or shortened peptides or polypeptides which can beroutinely obtained and tested by one of ordinary skill in the art usingthe teachings presented in the prior art.

The novel amino acid sequences disclosed in the present patentapplication can be used to provide different kind of reagents andmolecules. Examples of these compounds are binding proteins orantibodies that can be identified using their full sequence or specificfragments, such as antigenic determinants. Peptide libraries can be usedin known methods (Tribbick G, 2002) for screening and characterizingantibodies or other proteins binding the claimed amino acid sequences,and for identifying alternative forms of such polypeptides havingsimilar binding properties.

The present patent application discloses also fusion proteins comprisingany of the polypepudes described above. These polypeptides shouldcontain protein sequence heterologous to the one disclosed In thepresent patent application, without significatively impairing thechemotactic activity and possibly providing additional properties.Examples of such properties are an easier purification procedure, alonger lasting half-life in body fluids, an additional binding moiety,the maturation by means of an endoproteolytic digestion, orextracellular localization. This latter feature is of particularimportance for defining a specific group of fusion or chimeric proteinsincluded in the above definition since it allows the claimed moleculesto be localized in the space where not only isolation and purificationof these polypeptides is facilitated, but also where generallychemokines and their receptor interact.

Design of the moieties, ligands, and linkers, as well methods andstrategies for the construction, purification, detection and use offusion proteins are disclosed in the literature (Nilsson J et al., 1997;Methods Enzymol, Vol. 326-328, Academic Press, 2000). The preferredprotein sequences that can be comprised in the fusion proteins of theinvention belong to these protein sequences: membrane-bound protein,immunoglobulin constant region, multimerization domains, extracellularproteins, signal peptide-containing proteins, export signal-containingproteins. Features of these sequences and their specific uses aredisclosed in a detailed manner, for example, for albumin fusion proteins(WO 01/77137), fusion proteins including multimerization domain (WO01/02440, WO 00/24782, WO 94/10308, WO 97/30161), immuno-conjugates(Garnett M C, 2001), or fusion protein including sequences allowing thepurification of the recombinant products by affinity chromatography(Constans A, 2002; Burgess R R and Thompson N E, 2002; Lowe C R et al.,2001; Sheibani N, 1999).

Several studies on structure-activity features of chemokines indicatethat these proteins bind and activate receptors by making use of theamino-terminal region. Proteolytic digestion, mutagenesis, or chemicalmodifications directed to amino acids in this region can generatecompounds having antagonistic activity (Loetscher P and Clark-Lewis I,2001; Lambeir A et al., 2001, Proost P et al., 2001). Thus, antagonisticmolecules resulting from specific modifications (deletions,non-conservative substitutions, addition of chemical groups) of one ormore residues in the amino-terminal region or in other regions of thecorresponding chemokine are considered having therapeutic potential forinflammatory and autoimmune diseases (WO 02/28419; WO 00/27880; WO99/33989; Schwarz M K and Wells T, 1999). Therefore, a further object ofthe present patent application is represented by such kind ofantagonists generated by modifying the polypeptides of the invention.

The polypeptides of the invention can be used to generate andcharacterize ligands binding specifically to them. These molecules canbe natural or artificial, very different from the chemical point of view(binding proteins, antibodies, molecularly imprinted polymers), and canbe produced by applying the teachings in the art (WO 02/74938; Kuroiwa Yet al., 2002; Haupt K, 2002; van Dijk M A and van de Winkel J G, 2001;Gavilondo J V and Larrick J W, 2000). Such ligands can antagonize orinhibit the chemotactic activity of the polypeptide against which theyhave been generated. In particular, common and efficient ligands arerepresented by extracellular domain of a membrane-bound protein orantibodies, which can be in the form monoclonal, polyclonal, humanizedantibody, or an antigen-binding fragment.

The polypeptides and the polypeptide-based derived reagents describedabove can be in alternative forms, according to the desired method ofuse and/or production, such as active conjugates or complexes with amolecule chosen amongst radioactive labels, fluorescent labels, biotin,or cytotoxic agents.

Specific molecules, such as peptide mimetics, can be also designed onthe sequence and/or the structure of a polypeptde of the invention.Peptide mimetics (also called peptidomimetics) are peptides chemicallymodified at the level of amino acid side chains, of amino acidchirality, and/or of the peptide backbone. These alterations areintended to provide agonists or antagonists of the polypeptdes of theinvention with improved preparation, potency and/or pharmaco kineticsfeatures.

For example, when the peptide is susceptible to cleavage by peptidasesfollowing injection into the subject is a problem, replacement of aparticularly sensitive peptide bond with a non-cleavable peptide mimeticcan provide a peptide more stable and thus more useful as a therapeutic.Similarly, the replacement of an L-amino acid residue is a standard wayof rendering the peptide less sensitive to proteolysis, and finally moresimilar to organic compounds other than peptides. Also useful areamino-terminal blocking groups such as t-butyloxycarbonyl, acetyl,theyl, succinyl, methoxysuccinyl, suberyl, adipyl, azelayl, dansyl,benzyloxycarbonyl, fluorenylmethoxycarbonyl, methoxyazelayl,methoxyadipyl, methoxysuberyl, and 2,4-dinitrophenyl. Many othermodifications providing increased potency, prolonged activity, easinessof purification, and/or increased half-life are disclosed in the priorart (WO 02/10195; Villain M et al., 2001).

Preferred alternative, synonymous groups for amino acids derivativesincluded in peptide mimetics are those defined in Table II. Anon-exhaustive list of amino acid derivatives also includeaminoisobutyric acid (Aib), hydroxyproline (Hyp),1,2,3,4-tetrahydro-isoquinoline-3-COOH, indoline-2carboxylic acid,4-difluoro-proline, L-thiazolidine-4-carboxylic acid, L-homoproline,3,4-dehydro-proline, 3,4-dihydroxy-phenylalanine, cyclohexyl-glycine,and phenylglycine.

By “amino acid derivative” is intended an amino acid or amino acid-likechemical entity other than one of the 20 genetically encoded naturallyoccurring amino acids. In particular, the amino acid derivative maycontain substituted or non-substituted, linear, branched, or cyclicalkyl moieties, and may include one or more heteroatoms. The amino acidderivatives can be made de novo or obtained from commercial sources(Calbiochem-Novabiochem AG, Switzerland; Bachem, USA).

Various methodologies for incorporating unnatural amino acidsderivatives into proteins, using both in vitro and in vivo translationsystems, to probe and/or improve protein structure and function aredisclosed in the literature (Dougherty D A, 2000). Techniques for thesynthesis and the development of peptide mimetics, as well asnon-peptide mimetics, are also well known in the art (Golebiowski A etal., 2001; Hruby V J and Balse P M, 2000; Sawyer T K, in “StructureBased Drug Design”, edited by Veerapandian P, Marcel Dekker Inc., pg.557-663, 1997).

Another object of the present invention are isolated nucleic acidsencoding for the polypeptides of the invention having chemotacticactivity, the polypeptides binding to an antibody or a binding proteingenerated against them, the corresponding fusion proteins, or mutantshaving antagonistic activity as disclosed above. Preferably, thesenucleic acids should comprise a DNA sequence selected from the groupconsisting of SEQ ID NO: 1, 3, 5, 7, 9, 11, 13, or 15, or the complementof said DNA sequences.

Alternatively, the nucleic acids of the invention should hybridize underhigh stringency conditions, or exhibits at least about 85% identity overa stretch of at least about 30 nucleotides, with a nucleic acid selectedfrom the group consisting of SEQ ID NO: 1, 3, 5, 7, 9, 11, 13, or 15 ora complement of said DNA sequences. A further object of the presentinvention is therefore the polypeptides encoded by these purifiednucleic acids.

The wording “high stringency conditions” refers to conditions in ahybridization reaction that facilitate the association of very similarmolecules and consist in the overnight incubation at 60°-65° C. in asolution comprising 50% formamide, 5×SSC (150 m M NaCl, 15 m M trisodiumcitrate), 50 mM sodium phosphate (pH 76), 5× Denhardt's solution, 10%dextran sulphate, and 20 microgram/ml denatured, sheared salmon spermDNA, followed by washing the filters in 0.1×SSC at the same temperature.

These nucleic acids, including nucleotide sequences substantially thesame, can be comprised in plasmids, vectors and any other DNA constructwhich can be used for maintaining, modifying, introducing, or expressingthe encoded polypeptide. In particular, vectors wherein said nucleicacid molecule is operatively linked to expression control sequences canallow expression in prokaryotic or eukaryotic host cells of the encodedpolypeptide.

The wording “nucleotide sequences substantially the same” includes allother nucleic acid sequences that, by virtue of the degeneracy of thegenetic code, also code for the given amino acid sequences. In thissense, the literature provides indications on preferred or optimizedcodons for recombinant expression (Kane J F et al., 1995).

The nucleic acids and the vectors can be introduced into cells withdifferent purposes, generating transgenic cells and organisms. A processfor producing cells capable of expressing a polypeptide of the inventioncomprises genetically engineering cells with such vectors and nucleicacids.

In particular, host cells (e.g. bacterial cells) can be modified bytransformation for allowing the transient or stable expression of thepolypeptides encoded by the nucleic acids and the vectors of theinvention. Alternatively, said molecules can be used to generatetransgenic animal cells or non-human animals (by non-/homologousrecombination or by any other method allowing their stable integrationand maintenance), having a constitutive or inducible altered expressionlevels (i.e. enhanced or reduced) of the polypeptides of the invention,when the level is compared with the normal expression levels. Suchprecise modifications can be obtained by making use of the nucleic acidsof the inventions and of technologies associated, for example, to genetherapy (Meth. Enzymol., vol. 346, 2002) or to site-specificrecombinases (Kolb A F, 2002). Model systems based on the chemokine-likepolypeptides disclosed in the present patent application for thesystematic study of their function can be also generated by genetargeting into human cell lines (Bunz F, 2002).

The polypeptides of the invention can be prepared by any method known inthe art, including recombinant DNA-related technologies, and chemicalsynthesis technologies. In particular, a method for making a polypeptideof the invention may comprise culturing a host or transgenic cell asdescribed above under conditions in which the nucleic acid or vector isexpressed, and recovering the polypeptide encoded by said nucleic acidor vector from the culture. For example, when the vector expresses thepolypeptide as a fusion protein with an extracellular or signal-peptidecontaining proteins, the recombinant product can be secreted in theextracellular space, and can be more easily collected and purified fromcultured cells in view of further processing or, alternatively, thecells can be directly used or administered.

The DNA sequence coding for the proteins of the invention can beinserted and ligated into a suitable episomal or non-/homologouslyintegrating vectors, which can be introduced in the appropriate hostcells by any suitable means (transformation, transfection, conjugation,protoplast fusion, electroporation, calcium phosphate-precipitation,direct microinjection, etc.). Factors of importance in selecting aparticular plasmid or viral vector include: the ease with whichrecipient cells that contain the vector, may be recognized and selectedfrom those recipient cells which do not contain the vector; the numberof copies of the vector which are desired in a particular host; andwhether it is desirable to be able to “shuttle” the vector between hostcells of different species.

The vectors should allow the expression of the isolated or fusionprotein including the polypeptide of the invention by prokaryotic oreukaryotic host cells under the control of transcriptionalinitiation/termination regulatory sequences, which are chosen to beconstitutively active or inducible in said cell. A cell linesubstantially enriched in such cells can be then isolated to provide astable cell line.

Different transcriptional and translational regulatory sequences may beemployed for eukaryotic hosts (e.g. yeasts, insect, plant, or mammaliancells), depending on the nature of the host. They may be derived formviral sources, such as adenovirus, bovine papilloma virus, Simian virusor the like, where the regulatory signals are associated with aparticular gene which has a high level of expression. Examples are theTK promoter of the Herpes virus, the SV40 early promoter, the yeast GAL4gene promoter, etc. Transcriptional initiation regulatory signals may beselected which allow for repression and activation, so that expressionof the genes can be modulated. The cells stably transformed by theintroduced DNA can be selected by introducing one or more markersallowing the selection of host cells that contain the expression vector.The marker may also provide for phototrophy to an auxotropic host,biocide resistance, e.g. antibiotics, or heavy metals such as copper, orthe like. The selectable marker gene can either be directly linked tothe DNA gene sequences to be expressed, or introduced into the same cellby co-transfection.

Host cells may be either prokaryotic or eukaryotic. Preferred areeukaryotic hosts, e.g. mammalian cells, such as human, monkey, mouse,and Chinese Hamster Ovary (CHO) cells, because they providepost-translational modifications to proteins, including correct foldingand glycosylation. Also yeast cells can carry out post-translationalpeptide modifications including glycosylation. A number of recombinantDNA strategies exist which utilize strong promoter sequences and highcopy number of plasmids that can be utilized for production of thedesired proteins in yeast. Yeast recognizes leader sequences in clonedmammalian gene products and secretes peptides bearing leader sequences(i.e., pre-peptides).

The above mentioned embodiments of the invention can be achieved bycombining the disclosure provided by the present patent application onthe sequence of novel chemokine-like polypeptides with the knowledge ofcommon molecular biology techniques.

Many books and reviews provides teachings on how to clone and producerecombinant proteins using vectors and prokaryotic or eukaryotic hostcells, such as some titles in the series “A Practical Approach”published by Oxford University Press (“DNA Cloning 2: ExpressionSystems”, 1995; “DNA Cloning 4: Mammalian Systems”, 1996; “ProteinExpression”, 1999; “Protein Purification Techniques”, 2001).

Moreover, updated and more focused literature provides an overview ofthe technologies for expressing polypeptides in a high-throughput manner(Chambers S P, 2002; Coleman T A, et al., 1997), of the cell systems andthe processes used industrially for the large-scale production ofrecombinant proteins having therapeutic applications (Andersen D C andKrummen L, 2002; Chu L and Robinson D K, 2001), and of alternativeeukaryotic expression systems for expressing the polypeptide ofinterest, which may have considerable potential for the economicproduction of the desired protein, such the ones based on transgenicplants (Giddings G, 2001) or the yeast Pichia pastoris (Lin Cereghino GP et al., 2002). Recombinant protein products can be rapidly monitoredwith various analytical technologies during purification to verify theamount and the quantity of the expressed polypeptides (Baker K N et al.,2002), as well as to check if there Is problem of bioequivalence andimmunogenicity (Schellekens H, 2002; Gendel S M, 2002).

Totally synthetic chemokines are disclosed in the literature (Brown A etal., 1996), and many examples of chemical synthesis technologies, whichcan be effectively applied for the chemokine-like polypeptides of theinvention given their short length, are available in the literature, assolid phase or liquid phase synthesis technologies. for example, theamino acid corresponding to the carboxy-terminus of the peptide to besynthetized is bound to a support which is insoluble in organicsolvents, and by alternate repetition of reactions, one wherein aminoacids with their amino groups and side chain functional groups protectedwith appropriate protective groups are condensed one by one in orderfrom the carboxy-terminus to the amino-terminus, and one where the aminoacids bound to the resin or the protective group of the amino groups ofthe peptides are released, the peptide chain is thus extended in thismanner.

Solid phase synthesis methods are largely classified by the tBoc methodand the Fmoc method, depending on the type of protective group used.Typically used protective groups include tBoc (t-butoxycarbonyl), Cl-Z(2-chlorobenzyloxycarbonyl), Br-Z (2-bromobenzyloxycarbonyl), Bzl(benzyl), Fmoc (9-fluorenylmethoxycarbonyl), Mbh(4,4′-dimethoxydibenzhydryl), Mtr(4-methoxy-2,3,6-trimethylbenzenesulphonyl), Trt (trityl), Tos (tosyl),Z (benzyloxycarbonyl) and Cl2-Bzl (2,6-dichlorobenzyl) for the aminogroups; NO2 (nitro) and Pmc (2,2,5,7,8-pentamethylchromane-6-sulphonyl)for the guanidino groups); and tBu (t-butyl) for the hydroxyl groups).After synthesis of the desired peptide, it is subjected to thede-protection reaction and cut out from the solid support. Such peptidecutting reaction may be carried with hydrogen fluoride ortrifluoromethane sulfonic acid for the Boc method, and with TFA for theFmoc method.

The purification of the polypeptides of the invention can be carried outby any one of the methods known for this purpose, i.e. any conventionalprocedure involving extraction, precipitation, chromatography,electrophoresis, or the like. A further purification procedure that maybe used in preference for purifying the protein of the invention isaffinity chromatography using monoclonal antibodies or affinity groups,which bind the target protein and which are produced and immobilized ona gel matrix contained within a column. Impure preparations containingthe proteins are passed through the column. The protein will be bound tothe column by heparin or by the specific antibody while the impuritieswill pass through. After washing, the protein is eluted from the gel bya change in pH or ionic strength. Alternatively, HPLC (High PerformanceLiquid Chromatography) can be used. The elution can be carried using awater-acetonitrile-based solvent commonly employed for proteinpurification.

The disclosure of the novel polypeptides of the invention, and thereagents disclosed in connection to them (antibodies, nucleic acids,cells) allows also to screen and characterize compounds that enhance orreduce their expression level into a cell or in an animal. Examples ofcompounds that can reduce or block the expression of the chemokine-likepolypeptides are antisense oligonucleotides (Stein C A, 2001) or smallinterfering, double stranded RNA molecules that can trigger RNAinterference-mediated silencing (Paddison P J et al., 2002; Lewis D L etal., 2002). These compounds are intended as antagonists (in addition tothe ones above described in connection to mutants and ligands) in thecontext of the possible mechanism of antagonism for blockingcytokine/chemokine-controlled pathways as defined in the literature(Choy E H and Panayi G S, 2001; Dower S K, 2000).

“Oligonucleotides” refers to either a single strandedpolydeoxynucleotide or two complementary polydeoxynucleotide strandsthat may be chemically synthesized. Such synthetic oligonucleotides haveno 5′ phosphate and thus will not ligate to another oligonucleotidewithout adding a phosphate with an ATP in the presence of a kinase. Asynthetic oligonucleotide will ligate to a fragment that has not beendephosphorylated.

The invention includes purified preparations of the compounds of theinvention (polypeptides, nucleic acids, cells, etc.). Purifiedpreparations, as used herein, refers to the preparations containing atleast 1%, preferably at least 5%, by dry weight of the compound of theinvention.

The present patent application discloses a series of novelchemokine-like polypeptides and of related reagents having severalpossible applications. In particular, whenever an increase in thechemotactic activity of a polypeptide of the invention is desirable inthe therapy or in the prevention of a disease, reagents such as thedisclosed chemokine-like polypeptides, the corresponding fusion proteinsand peptide mimetics, the encoding nucleic acids, the expressing cells,or the compounds enhancing their expression can be used.

Therefore, the present invention discloses pharmaceutical compositionsfor the treatment or prevention of diseases needing an increase in thechemotactic activity of a polypeptide of the invention, which containone of the disclosed chemokine-like polypeptides, the correspondingfusion proteins and peptide mimetics, the encoding nucleic acids, theexpressing cells, or the compounds enhancing their expression, as activeingredient.

The process for the preparation of these pharmaceutical compositionscomprises combining the disclosed chemokine-like polypeptides, thecorresponding fusion proteins and peptide mimetics, the encoding nucleicacids, the expressing cells, or the compounds enhancing theirexpression, together with a pharmaceutically acceptable carrier.

Methods for the treatment or prevention of diseases needing an increasein the chemotactic activity of a polypeptide of the invention, comprisethe administration of a therapeutically effective amount of thedisclosed chemokine-like polypeptides, the corresponding fusion proteinsand peptide mimetics, the encoding nucleic acids, the expressing cells,or the compounds enhancing their expression.

Amongst the reagents disclosed in the present patent application, theligands, the antagonists or the compounds reducing the expression or theactivity of polypeptides of the invention have several applications, andin particular they can be used in the therapy or in the diagnosis of adisease associated to the excessive chemotactic activity of apolypeptide of the invention.

Therefore, the present invention discloses pharmaceutical compositionsfor the treatment or prevention of diseases associated to the excessivechemotactic activity of a polypeptide of the invention, which containone of the ligands, antagonists, or compounds reducing the expression orthe activity of such poly peptides, as active ingredient.

The process for the preparation of these pharmaceutical compositionscomprises combining the ligand, the antagonist, or the compound,together with a pharmaceutically acceptable carrier.

Methods for the treatment or prevention of diseases associated to theexcessive chemotactic activity of the polypeptide of the inventioncomprise the administration of a therapeutically effective amount of theantagonist, the ligand or of the compound.

The pharmaceutical compositions of the invention may contain, inaddition to chemokine-like polypeptide or to the related reagent,suitable pharmaceutically acceptable carriers, biologically compatiblevehicles and additives which are suitable for administration to ananimal (for example, physiological saline) and eventually comprisingauxiliaries (like excipients, stabilizers, adjuvants, or diluents) whichfacilitate the processing of the active compound into preparations whichcan be used pharmaceutically.

The pharmaceutical compositions may be formulated in any acceptable wayto meet the needs of the mode of administration. For example, ofbiomaterials, sugar-macromolecule conjugates, hydrogels, polyethyleneglycol and other natural or synthetic polymers can be used for improvingthe active ingredients in terms of drug delivery efficacy. Technologiesand models to validate a specific mode of administration are disclosedin literature (Davis B G and Robinson M A, 2002; Gupta P et al., 2002;Luo B and Prestwich G D, 2001; Cleland J L et al., 2001; Pillai O andPanchagnula R, 2001).

Polymers suitable for these purposes are biocompatible, namely, they arenon-toxic to biological systems, and many such polymers are known. Suchpolymers may be hydrophobic or hydrophilic in nature, biodegradable,non-biodegradable, or a combination thereof. These polymers includenatural polymers (such as collagen, gelatin, cellulose, hyaluronicacid), as well as synthetic polymers (such as polyesters,polyorthoesters, polyanhydrides). Examples of hydrophobic non-degradablepolymers include polydimethyl siloxanes, polyurethanes,polytetrafluoroethylenes, polyethylenes, polyvinyl chlorides, andpolymethyl methaerylates. Examples of hydrophilic non-degradablepolymers include poly(2-hydroxyethyl methacrylate), polyvinyl alcohol,poly(N-vinyl pyrrolidone), polyalkylenes, polyacrylamide, and copolymersthereof. Preferred polymers comprise as a sequential repeat unitethylene oxide, such as polyethylene glycol (PEG).

Any accepted mode of administration can be used and determined by thoseskilled in the art to establish the desired blood levels of the activeingredients. For example, administration may be by various parenteralroutes such as subcutaneous, intravenous, intradermal, intramuscular,intraperitoneal, intranasal, transdermal, oral, or buccal routes. Thepharmaceutical compositions of the present invention can also beadministered in sustained or controlled release dosage forms, includingdepot injections, osmotic pumps, and the like, for the prolongedadministration of the polypeptide at a predetermined rate, preferably inunit dosage forms suitable for single administration of precise dosages.

Parenteral administration can be by bolus injection or by gradualperfusion over time. Preparations for parenteral administration includesterile aqueous or non-aqueous solutions, suspensions, and emulsions,which may contain auxiliary agents or excipients known in the art, andcan be prepared according to routine methods. In addition, suspension ofthe active compounds as appropriate oily injection suspensions may beadministered. Suitable lipophilic solvents or vehicles include fattyoils, for example, sesame oil, or synthetic fatty acid esters, forexample, sesame oil, or synthetic fatty acid esters, for example, ethyloleate or triglycerides. Aqueous injection suspensions that may containsubstances increasing the viscosity of the suspension include, forexample, sodium carboxymethyl cellulose, sorbitol, and/or dextran.Optionally, the suspension may also contain stabilizers. Pharmaceuticalcompositions include suitable solutions for administration by injection,and contain from about 0.01 to 99.99 percent, preferably from about 20to 75 percent of active compound together with the excipient.

The wording “therapeutically effective amount” refers to an amount ofthe active ingredients that is sufficient to affect the course and theseverity of the disease, leading to the reduction or remission of suchpathology. The effective amount will depend on the route ofadministration and the condition of the patient.

The wording “pharmaceutically acceptable” is meant to encompass anycarrier, which does not interfere with the effectiveness of thebiological activity of the active ingredient and that is not toxic tothe host to which is administered. For example, for parenteraladministration, the above active ingredients may be formulated in unitdosage form for injection in vehicles such as saline, dextrose solution,serum albumin and Ringer's solution. Carriers can be selected also fromstarch, cellulose, talc, glucose, lactose, sucrose, gelatin, malt, rice,flour, chalk, silica gel, magnesium stearate, sodium stearate, glycerolmonostearate, sodium chloride, dried skim milk, glycerol, propyleneglycol, water, ethanol, and the various oils, including those ofpetroleum, animal, vegetable or synthetic origin (peanut oil, soybeanoil, mineral oil, sesame oil).

It is understood that the dosage administered will be dependent upon theage, sex, health, and weight of the recipient, kind of concurrenttreatment, if any, frequency of treatment, and the nature of the effectdesired. The dosage will be tailored to the individual subject, as isunderstood and determinable by one of skill in the art. The total doserequired for each treatment may be administered by multiple doses or ina single dose. The pharmaceutical composition of the present inventionmay be administered alone or in conjunction with other therapeuticsdirected to the condition, or directed to other symptoms of thecondition. Usually a daily dosage of active ingredient is comprisedbetween 0.01 to 100 milligrams per kilogram of body weight per day.Ordinarily 1 to 40 milligrams per kilogram per day given in divideddoses or in sustained release form is effective to obtain the desiredresults. Second or subsequent administrations can be performed at adosage, which is the same, less than, or greater than the initial orprevious dose administered to the individual.

Apart from the methods having a therapeutic or a production purpose,several other methods can make use of the chemokine-like polypeptidesand of the related reagents disclosed in the present patent application.

In a first example, a method for screening candidate compounds effectiveto treat a disease related to a chemokine-like polypeptides of theinvention, comprises:

-   -   (a) contacting host cells expressing such polypeptide,        transgenic non-human animals, or transgenic animal cells having        enhanced or reduced expression levels of the polypeptide, with a        candidate compound; and    -   (b) determining the effect of the compound on the animal or on        the cell.

In a second example, a method for identifying a candidate compound as anantagonist/inhibitor or agonist/activator of a polypeptide of theinvention comprises:

-   -   (a) contacting the polypeptide, the compound, and a mammalian        cell or a mammallian cell membrane; and    -   (b) measuring whether the molecule blocks or enhances the        interaction of the polypeptide, or the response that results        from such interaction, with the mammalian cell or the mammalian        cell membrane.

In a third example, methods for determining the activity and/or thepresence of the polypeptide of the invention in a sample, can detecteither the polypeptide or the encoding RNA/DNA. Thus, such a methodcomprises:

-   -   (a) providing a protein-containing sample;    -   (b) contacting said sample with a ligand of the invention; and    -   (c) determining the presence of said ligand bound to said        polypeptide, thereby determining the activity and/or the        presence of polypeptide in said sample.

Alternatively, the method comprises:

-   -   (a) providing a nucleic acids-containing sample;    -   (b) contacting said sample with a nucleic acid of the invention;        and    -   (c) determining the hybridizauon of said nucleic acid with a        nucleic acid into the sample, thereby determining the presence        of the nucleic acid in the sample.

In this context, primer sequences containing the sequences SEQ ID NO:17-28 (Table III) can be used as well for determining the presence orthe amount of a transcript or of a nucleic acid encoding a polypeptideof invention in a sample by means of Polymerase Chain Reactionamplification.

A further object of the present invention are kits for measuring theactivity and/or the presence of chemokine-like polypeptide of theinvention in a sample comprising one or more of the reagents disclosedin the present patent application: a chemokine-like polypeptide of theinvention, an antagonist, ligand or peptide mimetic, an isolated nucleicacid or the vector, a pharmaceutical composition, an expressing cell, acompound increasing or decreasing the expression levels, and/or primersequences containing the sequences SEQ ID NO: 17-28.

Those kits can be used for in vitro diagnostic or screenings methods,and their actual composition should be adapted to the specific format ofthe sample (e.g. biological sample tissue from a patient), and themolecular species to be measured. For example, if it is desired tomeasure the concentration of the chemokine-like polypeptide, the kit maycontain an antibody and the corresponding protein in a purified form tocompare the signal obtained in Western blot. Alternatively, if it isdesired to measure the concentration of the transcript for thechemokine-like polypeptide, the kit may contain a specific nucleic acidprobe designed on the corresponding ORF sequence, or may be in the formof nucleic acid array containing such probe, or the primer sequencesdisclosed as SEQ ID NO: 17-28 (Table III). The kits can be also in theform of protein-, peptide mimetic-, or cell-based microarrays (Templin MF et al., 2002; Pellois J P et al., 2002; Blagoev B and Pandey A, 2001),allowing high-throughput proteomics studies, by making use of theproteins, peptide mimetics and cells disclosed in the present patentapplication.

The novel chemokine-like polypeptides of the invention and the relatedreagents that may be useful, as active ingredients in pharmaceuticalcompositions appropriately formulated, in the treatment or prevention ofdiseases such as cell proliferative disorders, autoimmune/inflammatorydisorders, cardiovascular disorders, neurological disorders,developmental disorders, metabolic disorder, infections and otherpathological conditions. In particular, given the known properties ofchemokines, the disclosed polypeptides and reagents should addressconditions involving abnormal or defective cell migration.Non-limitative examples of such conditions are the following: arthritis,rheumatoid arthritis (RA), psoriatic arthritis, osteoarthritis, systemiclupus erythematosus (SLE), systemic sclerosis, scleroderma,polymyositis, glomerulonephritis, fibrosis, lung fibrosis andinflammation, allergic or hypersensitvity diseases, dermatitis, asthma,chronic obstructive pulmonary disease (COPD), inflammatory bowel disease(IBD), Crohn's diseases, ulcerative colitis, multiple sclerosis, septicshock, HIV infection, transplant rejection, wound healing, metastasis,endometriosis, hepatitis, liver fibrosis, cancer, analgesia, andvascular inflammation related to atherosclerosis.

The therapeutic applications of the polypeptides of the invention and ofthe related reagents can be evaluated (in terms or safety,pharmacokinetics and efficacy) by the means of the in vivo or in vitroassays making use of animal cell, tissues and models (Coleman R A etal., 2001; Li A P, 2001; Methods Mol. Biol vol. 138, “ChemokinesProtocols”, edited by Proudfoot A et al., Humana Press Inc., 2000;Methods Enzymol, vol. 287 and 288, Academic Press, 1997), or by themeans of in silico, computational approaches (Johnson D E and Wolfgang GH, 2000), known for the validation of chemokines and other biologicalproducts during drug discovery and preclinical development.

All publications, patents and patent applications cited herein areincorporated in full by reference for all purposes.

The invention will now be described with reference to the specificembodiments by means of the following Examples, which should not beconstrued as in any way limiting the present invention. The content ofthe description comprises all modifications and substitutions which canbe practiced by a person skilled in the art in light of the aboveteachings and, therefore, without extending beyond the meaning andpurpose of the claims.

EXAMPLES Example 1 Selection of Chemokine-Like Open Reading Frames(ORFS) from Human Genome

Perl (Practical Extraction and Report Language) is a programminglanguage having powerful pattern matching functions into large text datafiles allowing the extraction of information from genomic DNA sequences,starting from an alpha-numerical expression describing a definedconsensus sequence (Stein L D, 2001).

A Perl script was used to retrieve novel open reading frames (ORFs),having chemokine-like features, in a FASTA-formatted sequence filecontaining the NCBI genome (build 28). After translating the genomic DNAsequence into the six possible reading frames (3 forward and 3 reverse),each of these translated sequences was then tested for a match against apattern designed to detect to chemokine-like proteins, which waselaborated comparing multiple sequence alignments of known chemokines.The following pattern, fitting all the aligned sequences, was adopted:{M}-{X}₃₋₁₂-{L or I or V}₁₋₃—{X}₀₋₂-{L or I or V}₂₋₄—{X}₀₋₂-{L or I orV}₁₋₃—{X}₁₀₋₃₀—{C}—(X)₁₀₋₃₀—C—(X)₂₀₋₄₀—C—{X}₁₂₋₂₀—{C}—{X}₁₅₋₄₀ STOP

The letter(s) between brackets represented alternative amino acids (inone-letter code) which should be present the number of times indicatedin subscript characters. This expression, which describes the entirefamily of sequences on the basis of the respective positioning of theinitial methionine, hydrophobic residues, and conserved cysteines on thelinear sequence, can be transformed in Perl language as follows:M[ˆ\*]{3,12}[LIV]{1,3}[ˆ\*]{0,2}[LIV]{2,4}[ˆ\*]{0,2}[LIV]{1,3}[ˆ\*]{10,30}C[ˆ\*]{0,3}C[ˆ\*]{20,40}C[ˆ\*]{12,20}C[ˆ\*]{15,40}[\*]

A total of FASTA-formatted 7974 ORFs matching the pattern were comparedto known proteins present in protein databases (SwissProt/Trembl andDerwent GENESEQ) using a rapid searching program for local alignmentsbetween a query and a hit sequence based on Basic Local Alignment SearchTool (BLAST, BLASTX) and ClustalW algorithms (Altschul S F et al., 1990;Pearson W R and Miller W, 1992; Gish W and States D J, 1993). BLASTparameters used were: Comparison matrix=BLOSUM62; word length=3; .Evalue cutoff=10; Gap opening and extension=default; No filter.

The sequences obtained from this first screening were further selectedusing additional criteria. 2441 ORFs showing at least 70% of homologywith known proteins in protein databases were eliminated. The remaining5533 ORFs were filtered using 2 neural network-based algorithmsdeveloped for the prediction (probability at least 0.7) of a N-terminalsignal peptide and of an alpha helix secondary structure having at least5 amino acids within the C-terminal 30 amino acids (a hallmark of theIL8-like fold) with high confidence. The resulting 253 ORFs, which werepredicted as containing these features, were transformed in text formatand were compared to known chemokines, searching manually for the bestalignments. This further refinement, based on the qualitative assessmentof the alignments, led to the selection of eight chemokine-like encodingORFs for which all criteria for the prediction (sequence length,cysteine spacing, N-terminal signal sequence, C-terminal alpha helix)were fulfilled, making them comparable to known chemokines.

The DNA sequence GNSQ_(—)1754 (SEQ ID NO: 1), belonging to humanchromosome 13, contains an ORF encoding for the 98-amino acid longprotein sequence p1754 (SEQ ID NO: 2), which, according to theprediction, presents a 22-amino acid long signal sequence and an alphahelix covering the residues 70-79 (FIG. 1).

The DNA sequence GNSQ_(—)0711 (SEQ ID NO: 3), belonging to humanchromosome 16, contains an ORF encoding for the 109-amino acid longprotein sequence p0711 (SEQ ID NO: 4), which, according to theprediction, presents a 17-amino acid long signal sequence and an alphahelix covering the residues 98-106 (FIG. 2).

The DNA sequence GNSQ_(—)2882 (SEQ ID NO: 5), belonging to humanchromosome 6, contains an ORF encoding for the 107-amino acid longprotein sequence p2882 (SEQ ID NO: 6), which, according to theprediction, presents a 18-amino acid long signal sequence and an alphahelix covering the residues 96-104 (FIG. 3).

The DNA sequence GNSQ_(—)4711 (SEQ ID NO: 7), belonging to humanchromosome 3, contains an ORF encoding for the 102-amino acid longprotein sequence p4711 (SEQ ID NO: 8), which, according to theprediction, presents a 22-amino acid long signal sequence and an alphahelix covering the residues 83-97 (FIG. 4).

The DNA sequence GNSQ_(—)4320 (SEQ ID NO: 9), belonging to humanchromosome 3, contains an ORF encoding for the 101-amino acid longprotein sequence p4320 (SEQ ID NO: 10), which, according to theprediction, presents a 16-amino acid long signal sequence and an alphahelix covering the residues 90-98 (FIG. 5).

The DNA sequence GNSQ_(—)5008 (SEQ ID NO: 11), belonging to humanchromosome 12, contains an ORF encoding for the 112-amino acid longprotein sequence p5008 (SEQ ID NO: 12), which, according to theprediction, presents a 17-amino acid long signal sequence and an alphahelix covering the residues 95-109 (FIG. 6).

The DNA sequence GNSQ_(—)0210 (SEQ ID NO: 13), belonging to humanchromosome 7, contains an ORF encoding for the 127-amino acid longprotein sequence p0210 (SEQ ID NO: 14), which, according to theprediction, presents a 16-amino acid long signal sequence and an alphahelix covering the residues 94-113 (FIG. 7).

The DNA sequence GNSQ_(—)4922 (SEQ ID NO: 15), belonging to humanchromosome 10, contains an ORF encoding for the 91-amino acid longprotein sequence p4922 (SEQ ID NO: 14), which, according to theprediction, presents a 23-amino acid long signal sequence and an alphahelix covering the residues 67-74 (FIG. 8).

Amongst these sequences characterized as encoding chemokine-likepolypeptides, five of them (p1754, p0711, p2882, p0210, and p4922)present a central Cys-rich region in which the first two Cysteines areseparated by 1-3 amino acids, and can be compared with known C—X—Cchemokines (FIG. 9). The remaining three sequences (p4711, p4320, andp5008) present two adjacent Cysteines at the beginning of such region,and therefore can be compared with known C—C chemokines (FIG. 10).

Example 2 Cloning of the Novel Chemokine-Like ORFs from Human GenomicDNA

Six of the eight above-defined chemokine-like ORFs (GNSQ_(—)1754,GNSQ_(—)4922, GNSQ_(—)5008, GNSQ_(—)0210, GNSQ_(—)0711, andGNSQ_(—)4320) were first cloned from human genomic DNA into a cloningvector, and then transferred into an expression vector using PolymeraseChain Reaction (PCR), with pairs of forward/reverse primers specific foreach ORF (see arrows in FIGS. 1, 2, and 5-8).

The cloning primers (CL series; Table III), having a length comprisedbetween 19 and 25 bases, were designed for amplifying each ORF, usinghuman genomic DNA as template. The forward primers start from theinitial ATG or a few nucleotides before. The reverse primers arecomplementary to the 3′ end of the ORF, including the stop codon.

The PCR was performed by mixing the following components in eachORF-specific reaction (total volume of 50 μl in double-distilled water):

-   -   150 ng human genomic DNA (Clontech)    -   1.2 μM primers (0.6 μM each primer)    -   240 μM dNTP (Invitrogen)    -   0.5 μl AmpliTaq (2.5 Units; Applied Biosystems)    -   5 μl AmpliTaq buffer 10× (Applied Biosystems)

The PCR reactions were performed using an initial denaturing step if 94°C. for 2 minutes, followed by 30 cycles:

-   -   94° C. for 30 seconds    -   55° C. for 30 seconds    -   72° C. for 30 seconds

After a final elongation step of 72° C. for 10 minutes, the PCR productswere directly subcloned into the pCRII-TOPO vector using the TOPO™cloning system (Invitrogen), according to manufacturer's standardprotocol. The TOPO cloning system is a variation of the TA cloningsystem allowing the rapid cloning of PCR products, taking advantage fromthe fact that Taq polymerase leaves a single Adenosine at the 3′ end ofPCR products. Since the TOPO vector has single-stranded Thymineoverhangs, Topoisomerase I enzyme is able to join the T-ends of thevector to the A-overhangs of the PCR product, which can be used withoutany purification step.

The resulting plasmids (pCRTOPO-ORF series) were used to transform E.coli cells (TOP10F′, Invitrogen, supplied with the TOPO TA Cloning Kit),obtaining several clones for each ORF. Plasmid DNA was isolated using acommercial kit (WIZARD Plasmid Minipreps; Promega) and sequenced toverify the identity of the amplified and cloned sequence with theoriginally selected human genomic DNA sequence.

The plasmids containing the desired sequences were used in a furtherround of PCR reactions necessary for transferring the ORFs into theexpression vector pEAK12d (FIG. 11), which allows the expression of thecloned insert under the control of EF-1α promoter and in frame with a6-Histidine Tag sequence, using the Gateway cloning system (Invitrogen).

The expression vector pEAK12D was constructed by modifying pEAK12 (EdgeBiosystems). This vector was digested with HindIII and NotI, made bluntended with Klenow and dephosphorylated using calf-intestinal alkalinephosphatase. After dephosphorylation, the vector was ligated to bluntended Gateway reading frame cassette C (Gateway vector conversionsystem, Invitrogen cat no. 11828-019) that contains AttR recombinationsites flanking the ccdB gene (marker for negative selction ofnon-recombinant plasmids) and chloramphenicol resistance. The resultingplasmids were used to transform DB3.1 E. coli cells, which allowpropagation of vectors containing the ccdB gene. Miniprep DNA wasisolated from several of the resultant colonies and digested withAseI/EcoRI to identify clones yielding a 670 bp fragment, obtainableonly when the cassette had been inserted in the correct orientation. Theresultant plasmid was called pEAK12D.

Two series of primers (Table IV) were designed to add the ATTB1 andATTB2 recombination sites (necessary for the integration in theexpression vector) at the 5′ and 3′ end, respectively, of theORF-containing insert. In the first series of primers (EX1 series), theoriginal ORF-specific CL primers were modified by adding, at the 5′ end,the sequence AAGCAGGCTTCGCCACC (for forward primers) or GTGATGGTGATGGTG(for reverse primers, but after eliminating the nucleotidescomplementary to the stop codon). In the second series of primers (EX2series), the original ORF-specific CL primers were modified by adding,at the 5′ end, the sequence GGGGACAAGTTTGTACAAAAAAGCAGGCTTCGCCACC (forforward primers) or GGGGACCACTTTGTACAAGAAAGCTGGGTTTCAATGGTGATGGTGATGGTG(for reverse primers, but after eliminating the nucleotidescomplementary to the stop codon). These reverse primers contain thecodons for the 6-Histidine tag, which then results fused in frame withthe ORFs at their C-terminal end.

The PCR amplification was performed in 2 consecutive reactions. Thefirst one was performed by mixing the following components (total volume50 μl in double-distilled water):

-   -   25 ng pCRTOPO-ORF vector    -   5 mM dNTP (Invitrogen)    -   0.5 μl Pfx DNA polymerase (Invitrogen)    -   0.5 μl each EX1 primer (100 μM)    -   5 μl Pfx polymerase buffer 10× (Invitrogen)

The PCR reactions were performed using an initial denaturing step of 95°C. for 2 minutes, followed by 10 cycles:

-   -   94° C. for 15 seconds    -   68° C. for 30 seconds

The PCR products were purified using the Wizard PCR prep DNApurification system (Promega), and added as templates in a second PCRreaction including the following components (total volume 50 μl Indouble-distilled water):

-   -   10 μl purified PCR product    -   5 mM dNTP (Invitrogen)    -   0.5 μl Pfx DNA polymerase (Invitrogen)    -   0.5 μl each EX2 primer (100 μM)    -   5 μl Pfx polymerase buffer 10× (Invitrogen)

The PCR reactions were performed an initial denaturing step of 95° C.for 1 minute, followed by 4 cycles:

-   -   94° C. for 15 seconds    -   50° C. for 30 seconds    -   68° C. for 3 minutes 30 seconds

Then the following conditions were applied for 25 cycles:

-   -   94° C. for 15 seconds    -   55° C. for 30 seconds    -   68° C. for 3 minutes 30 seconds.

The DNA fragments resulting from the PCR reactions were purified asdescribed before and recombined into the pEAK12d vector using theGateway system.

First, the following 10 μl reactions were assembled: pDONR-201 (0.1μg/μl) 1.5 μl PCR product 5 μl BP buffer 2 μl BP enzyme mix 1.5 μl

After being incubated at room temperature for 1 hour, the reaction wasstopped by adding proteinase K (1 μl, 2 μg) and incubating at 37° C. forfurther 10 minutes.

An aliquot of this reaction (2 μl) was used for transforming E. colicells (strain DH10B) by electroporation. Plasmid DNA was prepared for 4clones for each ORF and used for parallel 10 μl recombination reactionscontaining: pEAK12d (0.1 μg/μl) 1.5 μl Plasmid DNA 1.5 μl ddH20 3.5 μlLR buffer 2 μl LR enzyme mix 1.5 μl

After being incubated at room temperature for 1 hour, the reaction wasstopped by adding proteinase K (1 μl, 2 μg) and incubating at 37° C. forfurther 10 minutes. An aliquot of this reaction (1 μl) was used fortransforming DH10B E. coli cells by electroporation. The clonescontaining the correct insert were identified first by performing colonyPCR on 3 colonies using the forward and reverse vector primers pEAK12dF1 (GCCAGCTTGGCACTTGATGT) and pEAK12d R1 (GATGGAGGTGGACGTGTCAG), thenconfirmed by sequencing the insert with the same primer.

Example 3 Expression and Purification of the 6-Histine-TaggedChemokine-Like Polypeptides in Mammalian Cells

Human Embryonic Kidney cells expressing the Epstein-Barr virus NuclearAntigen (HEK293-EBNA) were seeded in T225 flasks (50 ml at a density of2×10⁵ cells/ml) from 16 to 20 hours prior to transfection, which wasperformed using the cationic polymer reagent JetPEI™(PolyPlus-transfection; 2 μl/μg of plasmid DNA). For each flask, 113 μgof the ORF-specific pEAK12d plasmid, which were prepared using CsCl(Sambrook, J et al. “Molecular Cloning, a laboratory manual”; 2ndedition. 1989; Cold Spring Harbor Laboratory Press), were co-transfectedwith 2.3 μg of a plasmid acting as positive control since it expressesGreen Fluorescent Protein (GFP). The plasmids, diluted in 230 μl ofJetPEI™ solution, were added to 4.6 ml of NaCl 150 mM, vortexed andincubated for 30 minutes at room temperature. This transfection mix wasthen added to the T225 flask and incubated at 37° C. for 6 days. Analiquot of the cultures was then exposed to UV irradiation to check thetransfection efficiency by evaluating GFP fluorescence.

Culture medium from HEK293-EBNA cells transfected with the same plasmidswere pooled and 100 ml of the medium were diluted to 200 ml with 100 mlof ice-cold buffer A (50 mM NaH₂PO₄; 600 mM NaCl; 8.7% (w/v) glycerol,pH 7.5), which is the same buffer used for equilibrating the affinitycolumn on which His-tagged proteins were subsequently immobilized andeluted. The solution was filtered through a 0.22 μm sterile filter(Millipore) and kept at 4° C. in 250 ml sterile square media bottlesuntil further processing.

Two consecutive chromatography procedures were applied to the samples at4° C. using an HPLC-based system (Perfusion Chromatography™, PerSeptiveBiosystems) including a VISION workstation (BioCAD™ series), POROS™chromatographic media, and an external 250 ml-sample loader (Labomatic).

In the first chromatography step, a Ni-metal affinity column (0.83 ml,POROS 20 MC) was first regenerated with 30 column volumes of EDTAsolution (100 mM EDTA; 1 M NaCl; pH 8.0), and then recharged with Niions through washing with 15 column volumes of the Ni solution (100 mMNiSO₄). The column is subsequently washed with 10 column volumes ofbuffer A, 7 column volumes of buffer B (50 mM.NaH₂PO₄; 600 mM NaCl; 8.7%(w/v) glycerol, 400 mM; imidazole, pH 7.5), and finally equilibratedwith 15 column volumes of buffer A containing 15 mM imidazole. Thesample loader charged the protein-containing solution onto the Ni metalaffinity column at a flow rate of 10 ml/min. The column was then washedwith 12 column volumes of Buffer A, followed by 28 column volumes ofBuffer A containing a concentration of imidazole (20 mM) allowing theelution of contaminating proteins that are loosely attached to theNi-column. The His-tagged protein is finally eluted with 10 columnvolumes of Buffer B at a flow rate of 2 ml/min, collecting collected 1.6ml fractions.

In the second chromatography step, a gel-filtration column (10 ml G-255Sephadex) was regenerated with 2 ml of buffer D (137 mM NaCl; 2.7 mMKCl; 1.5 mM KH₂PO₄; 8 mM Na₂HPO₄; 1 M NaCl; pH 7.2), and thenequilibrated with 2 column volumes of buffer C (137 mM NaCl; 2.7 mM KCl;1.5 mM KH₂PO₄; 8 mM Na₂HPO₄; 20% (w/v) glycerol; pH 7.4) beforeinjecting the Ni-column peak fractions onto this column. The sample iseluted with buffer C and the desalted sample is recovered in 2.2 mlfractions. The peak fractions from the gel-filtration column werefiltered through a 0.22 μm sterile centrifugation filter (Millipore) andaliquots (20 μl) were analyzed in parallel on SDS-PAGE (4-12% NuPAGEgel; Novex) by Coomassie staining and by Western blot with antibodiesrecognizing Histidine tags. Protein concentrations were determined inthe samples that show detectable protein bands by Coomassie staining,using the BCA Protein Assay kit (Pierce) and Bovine Serum Albumin asstandard.

The gel for the Western blot analysis was electrotransferred to anitrocellulose membrane at 290 mA at 4° C. for 1 hour. The membrane isblocked with 5% milk powder in PBS (137 mM NaCl; 2.7 mM KCl; 1.5 mMKH₂PO₄; 8 mM Na₂HPO₄;pH 7.4), and subsequently incubated with a mixtureof 2 rabbit polyclonal anti-Histidine tag antibodies (G-18 and H-15, 0.2μg/ml each; Santa Cruz) at 4° C. overnight. After a further 1 hourincubation at room temperature, the membrane was washed with PBScontaining 0.1% Tween-20 (3×10 min), and then exposed to a secondaryHRP-conjugated anti-rabbit antibody (DAKO) at room temperature for 2hours. After washing in PBS containing 0.1% Tween-20 (3×10 minutes), theECL kit (Amersham Pharmacia) was used to detect the antibodiesimmobilized onto the membrane, comparing the film with the image of theCoomassie stained gel.

Example 4 Cell- and Animal-Based Assay for the Validation andCharacterization of the Chemokine-Like Polypeptides

Several assays have been developed for testing specificity, potency, andefficacy of chemokines using cell cultures or animal models, for examplein vitro chemotaxis assays (Proudfoot A E et al., 2001; Lust-NarasimhanM et al., 1995), or mouse ear swelling (Garrigue J L et al., 1994). Manyother assays and technologies for generating useful tools and products(antibodies, transgenic animals, radiolabeled proteins, etc.) have beendescribed in reviews and books dedicated to chemokines (Methods Mol.Biol vol. 138, “Chemokines Protocols”, edited by Proudfoot A I et al.,Humana Press Inc., 2000; Methods Enzymol, vol. 287 and 288, AcademicPress, 1997), and can be used to verify, in a more precise manner, thebiological activities of the chemokine-like polypeptides of theinvention and related reagents in connection with possible therapeuticor diagnostic methods and uses. TABLE I More Preferred Synonymous AminoAcid Synonymous Groups Groups Ser Gly, Ala, Ser, Thr, Pro Thr, Ser ArgAsn, Lys, Gln, Arg, His Arg, Lys, His Leu Phe, Ile, Val, Leu, Met Ile,Val, Leu, Met Pro Gly, Ala, Ser, Thr, Pro Pro Thr Gly, Ala, Ser, Thr,Pro Thr, Ser Ala Gly, Thr, Pro, Ala, Ser Gly, Ala Val Met, Phe, Ile,Leu, Val Met, Ile, Val, Leu Gly Ala, Thr, Pro, Ser, Gly Gly, Ala IlePhe, Ile, Val, Leu, Met Ile, Val, Leu, Met Phe Trp, Phe, Tyr Tyr, PheTyr Trp, Phe, Tyr Phe, Tyr Cys Ser, Thr, Cys Cys His Asn, Lys, Gln, Arg,His Arg, Lys, His Gln Glu, Asn, Asp, Gln Asn, Gln Asn Glu, Asn, Asp, GlnAsn, Gln Lys Asn, Lys, Gln, Arg, His Arg, Lys, His Asp Glu, Asn, Asp,Gln Asp, Glu Glu Glu, Asn, Asp, Gln Asp, Glu Met Phe, Ile, Val, Leu, MetIle, Val, Leu, Met Trp Trp, Phe, Tyr Trp

TABLE II Amino Acid Synonymous Groups Ser D-Ser, Thr, D-Thr, allo-Thr,Met, D-Met, Met(O), D-Met(O), L-Cys, D-Cys Arg D-Arg, Lys, D-Lys,homo-Arg, D-homo-Arg, Met, Ile, D-.Met, D-Ile, Orn, D-Orn Leu D-Leu,Val, D-Val, AdaA, AdaG, Leu, D-Leu, Met, D-Met Pro D-Pro,L-I-thioazolidine-4-carboxylic acid, D-or L-1-oxazolidine-4-carboxylicacid Thr D-Thr, Ser, D-Ser, allo-Thr, Met, D-Met, Met(O), D-Met(O), Val,D-Val Ala D-Ala, Gly, Aib, B-Ala, Acp, L-Cys, D-Cys Val D-Val, Leu,D-Leu, Ile, D-Ile, Met, D-Met, AdaA, AdaG Gly Ala, D-Ala, Pro, D-Pro,Aib, .beta.-Ala, Acp Ile D-Ile, Val, D-Val, AdaA, AdaG, Leu, D-Leu, Met,D-Met Phe D-Phe, Tyr, D-Thr, L-Dopa, His, D-His, Trp, D-Trp, Trans-3,4,or 5-phenylproline, AdaA, AdaG, cis-3,4, or 5-phenylproline, Bpa, D-BpaTyr D-Tyr, Phe, D-Phe, L-Dopa, His, D-His Cys D-Cys, S-Me-Cys, Met,D-Met, Thr, D-Thr Gln D-Gln, Asn, D-Asn, Glu, D-Glu, Asp, D-Asp AsnD-Asn, Asp, D-Asp, Glu, D-Glu, Gln, D-Gln Lys D-Lys, Arg, D-Arg,homo-Arg, D-homo-Arg, Met, D-Met, Ile, D-Ile, Orn, D-Orn Asp D-Asp,D-Asn, Asn, Glu, D-Glu, Gln, D-Gln Glu D-Glu, D-Asp, Asp, Asn, D-Asn,Gln, D-Gln Met D-Met, S—Me--Cys, Ile, D-Ile, Leu, D-Leu, Val, D-Val

TABLE III SEQ ID NO: NAME DIRECTION 5′→3′ SEQUENCE 17 CL_1754_5 ForwardATGAATGTCATTGTTTTACA 18 CL_1754_3 Reverse CTACCAACCTGTACAGCATG 19CL_4922_5 Forward CTGACTATGATGAGGGTGCT AAGGC 20 CL_4922_3 ReverseTCAAATTGCTGGGAAAGTTC TCAGG 21 CL_5008_5 Forward CATGATCTTTGGCCTGCTAA TC22 CL_5008_3 Reverse TTAAAGGGAAAGTAATAGGA G 23 CL_0210_5 ForwardCTATGGGCTTTGTTGTTCTA TG 24 CL_0210_3 Reverse TCAGAAAAATTCTAACAAAA TTG 25CL_0711_5 Forward ATGGTTATTCCACATCTTG 26 CL_0711_3 ReverseTCATCTCTGTTGCAGCAAAC G 27 CL_4320_5 Forward ATGTTATTTACTTTATTATT C 28CL_4320_3 Reverse TCACAGAAAAATCAAAGAGG

TABLE IV SEQ ID NO: NAME DIRECTION 5′→3′ SEQUENCE 29 EX1_1754_5 ForwardAAGCAGGCTTCGCCACCATGAATGTC ATTGTTTTACA 30 EX1_1754_3 ReverseGTGATGGTGATGGTGCCAACCTGTAC AGCATG 31 EX1_4922_5 ForwardAAGCAGGCTTCGCCACCCTGACTATG ATGAGGGTGCT AAGGC 32 EX1_4922_3 ReverseGTGATGGTGATGGTGAATTGCTGGGA AAGTTC TCAGG 33 EX1_5008_5 ForwardAAGCAGGCTTCGCCACC CATGATCT TTGGCCTGCTAA TC 34 EX1_5008_3 ReverseGTGATGGTGATGGTGAAGGGAAAGTA ATAGGA G 35 EX1_0210_5 ForwardAAGCAGGCTTCGCCACCCTATGGGCT TTGTTGTTCTA TG 36 EX1_0210_3 ReverseGTGATGGTGATGGTGGAAAAATTCTA ACAAAA TTG 37 EX1_0711_5 ForwardAAGCAGGCTTCGCCACCATGGTTATT CCACATCTTG 38 EX1_0711_3 ReverseGTGATGGTGATGGTGTCTCTGTTGCA GCAAACG 39 EX1_4320_5 ForwardAAGCAGGCTTCGCCACCATGTTATTT ACTTTATTATTC 40 EX1_4320_3 ReverseGTGAEGGTGATGGTGCAGAAAAATCA AAGAGG 41 EX2_1754_5 ForwardGGGGACAAGTTTGTACAAAAAAGCAG GCTTCGCCACCATGAATGTC ATTGT TTTACA 42EX2_1754_3 Reverse GGGGACCACTTTGTACAAGAAAGCTG GGTTTCAATGGTGATGGTGATGGTGCCAACCT GTACAGC 43 EX2_4922_5 Forward GGGGACAAGTTTGTACAAAAAAGCAGGCTTCGCCACC CTGACTATGATGAG GGTGCTAA 44 EX2_4922_3 ReverseGGGGACCACTTTGTACAAGAAAGCTG GGTTTCAATGGTGATGGTGATGGTGA ATTGCTCGGAAAGTTCTC 45 EX2_5008_5 Forward GGGGACAAGTTTGTACAAAAAAGCAG GCTTCGCCACCCATGATCTTTGGCC TGCTAATC 46 EX2_5008_3 Reverse GGGGACCACTTTGTACAAGAAAGCTGGGTTTCAATGGTGATGGTGATGGTGA AGGGAAAGTAATAGGAG 47 EX2_0210_5 ForwardGGGGACAAGTTTGTACAAAAAAGCAG GCTTCGCCACC CTATGGGCTTTGTT GTTCT 48EX2_0210_3 Reverse GGGGACCACTTTGTACAAGAAAGCTG GGTTTCAATGGTGATGGTGATGGTGGAAAAATTCTAACAAAA 49 EX2_0711_5 Forward GGGGACAAGTTTGTACAAAAAAGCAGGCTTCGCCACC ATGGTTATTCCACA TCTTG 50 EX2_0711_3 ReverseGGGGACCACTTTGTACAAGAAAGCTG GGTTTCAATGGTGATGGTGATGGTGT CTCTGTTGCAGCAAAC G51 EX2_4320_5 Forward GGGGACAAGTTTGTACAAAAAAGCAG GCTTCGCCACCATGTTATTTACTTT ATTATTC 52 EX2_4320_3 Reverse GGGGACCACTTTGTACAAGAAAGCTGGGTTTCAATGGTGATG GTGATGGTG CAGAAAAATCAAAGAGG

REFERENCES

-   Altschul S F et al., J Mol Biol, 215: 403-10, 1990.-   Andersen D C and Krummen L, Curr Opin Biotechnol, 13: 117-23, 2002.-   Baggiolini M et al., Annu Rev Immunol, 15: 675-705, 1997.-   Baggiolini M, J Intem Med, 250: 91-104, 2001.-   Baker K N et al., Trends Biotechnol, 20: 149-56, 2002.-   Blagoev B and Pandey A, Trends Biochem Sci, 26: 639 -41, 2001.-   Bock A, Science, 292: 453-4, 2001.-   Brown A et al., J Pept Sci, 2: 40-46, 1996.-   Browne M J, J Biotechnol, 78:247-250, 2000.-   Bunz F, Curr Opin Oncol, 14: 73-8, 2002.-   Burgess R R and Thompson N E, Curr Opin Biotechnol, 12: 450-4, 2001.-   Chambers S P, Drug Disc Today, 14: 759-765, 2002.-   Chantry D F et al., J Leukoc Biol, 64: 49-54, 1998.-   Choy E H and Panayi G S, N Engl J Med, 344: 907-16, 2001.-   Chu L and Robinson D K, Curr Opin Biotechnol, 13: 304-8, 2001.-   Cleland J L et al., Curr Opin Biotechnol, 12: 212-9, 2001.-   Coleman T A et al., Gene 190: 163-171, 1997.-   Coleman R A et al., Drug Discov Today, 6: 1116-1126, 2001.-   Constans A, The Scientist, 16: 37, 2002.-   Davis B G and Robinson M A, Curr Opin Drug Discov Devel, 5: 279-88,    2002.-   Dower S K, Nat Immunol, 1: 367-8, 2000.-   Dougherty D A, Curr Opin Chem Biol, 4: 645-52, 2000.-   Ehlert J E et al., J Immunol, 161: 4975-4982, 1998.-   Fernandez E J and Lolis E, Annu Rev Pharmacol Toxicol, 42: 469-499,    2002.-   Frederick M J and Clayman G L, Exp Rev Mol Med 2001,    http://www-ermm.cbcu.cam.ac.uk/reviews.htm.-   Garnett M C, Adv Drug Deliv Rev, 53: 171-216, 2001.-   Garrigue J L et al., Contact Dermattis, 30: 231-7, 1994.-   Gavilondo J V and Larrick J W, Biotechniques, 29: 128-136, 2000.-   Gendel S M, Ann N Y Acad Sci, 964: 87-98, 2002.-   Giddings G, Curr Opin Biotechnol, 12: 450-4, 2001.-   Gish W and States D J, Nat Genet, 3: 266-72, 1993.-   Godessart N and Kunkel S L, Curr Opin Immunol, 13: 670-675, 2001.-   Goleblowski A et al., Curr Opin Drug Discov Devel, 4: 428-34, 2001.-   Gupta P et al., Drug Discov Today, 7: 569-579, 2002.-   Haskell C A et al., Curr Opin Invest Drugs, 3: 399-455, 2002.-   Haupt K, Nat Biotechnol, 20: 884-885, 2002.-   Hruby V J and Balse P M, Curr Med Chem, 7: 945-70, 2000.-   Johnson D E and Wolfgang G H, Drug Discov Today, 5: 445-454, 2000.-   Kane J F, Curr Opin Biotechnol, 6: 494-500, 1995.-   Kolb A F, Cloning Stem Cells, 4: 65-80, 2002.-   Kuroiwa Y et al., Nat Biotechnol, 20: 889-94, 2002.-   Lambeir A, et al. J Biol Chem, 276: 29839-29845, 2001.-   Lewis D L, Nat Genet, 32: 107-8, 2002.-   Li A P, Drug Discov Today, 6: 357-366, 2001.-   Lin Cereghino G P et al., Curr Opin Biotechnol, 13: 329-332, 2001.-   Loetscher P and Clark-Lewis I, J Leukoc Biol, 69: 881-884, 2001.-   Lowe C R et al., J Biochem Biophys Methods, 49: 561-74, 2001.-   Lucas A D and Greaves D R, Exp Rev Mol Med 2001,    http://www-ermm.cbcu.cam.ac.uk/reviews.htm.-   Luo B and Prestwich G D, Exp Opin Ther Patents, 11: 1395-1410, 2001.-   Lusti-Narasimhan M et al., J Biol Chem, 270: 2716-21, 1995.-   Mulder N J and Apweiler R, Genome Biol, 3(1): REVIEWS2001, 2002-   Murphy L R et al., Protein Eng, 13: 149-52, 2000.-   Nilsson J et al., Protein Expr Purif, 11: 1-16, 1997.-   Nomiyama H et al., Genes Immun, 2: 110-113, 2001.-   Paddison P J, Proc Nati Acad Sci USA, 99: 1443-8, 2002.-   earson W R and Miller W, Methods Enzymol, 210: 575-601, 1992.-   Pellois J P et al., Nat Biotechnol, 20: 922-6, 2002.-   Pillai O and Panchagnula R, Cur Opin Chem Biol, 5: 447-451, 2001-   Proost P, et al. Blood, 98: 3554-3561, 2001.-   Proudfoot A., Immunol Rev, 177: 246-256, 2000.-   Proudfoot A et al., J Biol Chem 276: 10620-10626, 2001.-   Quinn-Senger K E et al., Curr Opin Chem Biol, 6: 418-26, 2002.-   Reape T J and Groot P H, Atherosclerosis, 147: 213-25, 1999.-   Rehm B H, Appl Microbiol Biotechnol, 57: 579-92, 2001.-   Robinson C R, Nat Biotechnol, 20: 879-880, 2002.-   Rogov S I and Nekrasov A N, Protein Eng, 14: 459-463, 2001.-   Rossi D et al., J.Immunol, 158: 1033-1036, 1997.-   Rossi D and Ziotnik A, Annu Rev Immunol, 18: 217-42, 2000.-   Schellekens H, Nat Rev Drug Discov, 1: 457-62, 2002-   Schwarz M K and Wells T N, Curr Opin Chem Biol, 3: 407-17, 1999.-   Shelbani N, Prep Biochem Biotechnol, 29: 77-90, 1999.-   Stein C A, J Clin Invest, 108: 641-4, 2001.-   Stein L D, Methods Biochem Anal, 43: 413-49, 2001.-   Stevanovic S, Nat Rev Cancer, 2: 514-20, 2002.-   Templin M F et al., Trends Biotechnol, 20: 160-6, 2002.-   Tribbick G, J Immunol Methods, 267: 27-35, 2002.-   van den Burg B and Eijsink V, Curr Opin Biotechnol, 13: 333-337,    2002.-   van Dijk M A and van de Winkel J G, Curr Opin Chem Biol, 5: 368-74,    2001.-   Villain M et al., Chem Biol, 8: 673-9, 2001.-   Wells T N and Peitsch M C. Methods Mol Biol, 138: 65-73, 2000.-   Yoshie O F et al., Adv Immunol, 78: 57-110, 2001.

1.-41. (canceled)
 42. A composition of matter comprising: a) an isolatedpolypeptide having chemotactic activity selected from the groupconsisting of: 1) the amino acid sequences SEQ ID NO: 2, 4, 6, 8, 10,12, 14, or 16; 2) the mature form of the polypeptides SEQ ID NO: 2, 4,6, 8, 10, 12, 14, or 16; 3) the polypeptides comprising theCysteine-rich region of SEQ ID NO: 2, 4, 6, 8, 10, 12, 14, or 16; 4) theactive variants of the amino acid sequence given by SEQ ID NO: 2, 4, 6,8, 10, 12, 14, or 16 wherein any amino acid specified in the chosensequence is non-conservatively substituted, provided that no more than15% of the amino acid residues in the sequence are so changed; 5) theactive fragments, precursors, salts, or derivatives of the amino acidsequences given in 1) to 4); 6) a naturally occurring allelic variant ofthe sequence given by SEQ ID NO: 2, 4, 6, 8, 10, 12, 14, or 16; 7) anaturally occurring allelic variant of the sequence given by SEQ ID NO:2, 4, 6, 8, 10, 12, 14, or 16, wherein the variant is the translation ofa single nucleotide polymorphism; 8) a polypeptide of any of 1) to 7),wherein the polypeptide binds specifically an antibody or a bindingprotein generated against SEQ ID NO: 2, 4, 6, 8, 10, 12, 14, or 16 or afragment thereof; 9) a fusion protein comprising a polypeptide accordingto any of 1) to 8); 10) a fusion protein comprising a polypeptideaccording to any of 1) to 8), wherein said proteins further comprise oneor more amino acid sequence belonging to these protein sequences:membrane-bound protein, immunoglobulin constant region, multimerizationdomains, extracellular proteins, signal peptide-containing proteins,export signal-containing proteins; 11) a polypeptide encoded by anucleic acid that hybridizes under high stringency conditions with anucleic acid selected from the group consisting of SEQ ID NO: 1, 3, 5,7, 9, 11, 13, or 15, or a complement of said DNA sequences or exhibitsat least about 85% identity over a stretch of at least about 30nucleotides, with a nucleic acid selected from the group consisting ofSEQ ID NO: 1, 3, 5, 7, 9, 11, 13, or 15, or a complement of said DNAsequences; b) an antagonist of a polypeptide of a1) to a8), wherein saidantagonist comprises an amino acid sequence resulting from themodification of one or more residues of said polypeptide; c) aligand: 1) that binds specifically to a polypeptide according to any oneof a1) to a8); 2) that binds specifically to a polypeptide according toany one of a1) to a8) and that antagonizes or inhibits the chemotacticactivity of a polypeptide according to any one of a1) to a8); 3) whichis a monoclonal antibody, a polyclonal antibody, a humanized antibody,an antigen binding fragment, or the extracellular domain of amembrane-bound protein; d) an isolated nucleic acid: 1) encodingpolypeptides of any of a1) to a8), said polypeptides having chemotacticactivity; 2) encoding the fusion proteins of a9) or a10); 3) encodingthe antagonists of b); 4) comprising a DNA sequence selected from thegroup consisting of SEQ ID NO: 1, 3, 5, 7, 9, 11, 13, or 15, or thecomplement of said DNA sequences; 5) that hybridizes under highstringency conditions with a nucleic acid selected from the groupconsisting of SEQ ID NO: 1, 3, 5, 7, 9, 11, 13, or 15, or a complementof said DNA sequences; or 6) exhibiting at least about 85% identity overa stretch of at least about 30 nucleotides, with a nucleic acid selectedfrom the group consisting of SEQ ID NO: 1, 3, 5, 7, 9, 11, 13, or 15, ora complement of said DNA sequences; e) a vector comprising a nucleicacid according to d); f) a peptide mimetic designed on the sequence orthe structure or both the sequence and structure of a polypeptideaccording to any one of a1) to a8); g) a host cell transformed with avector or a nucleic acid according to any of d) or e); h) a primercomprising SEQ ID NO: 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, or 28;i) a compound that enhances the expression level of a polypeptideaccording to any one a1) to a8); j) a compound that reduces theexpression level of a polypeptide according to any one a1) to a8); k) apharmaceutical composition comprising any one of a) to j) and apharmaceutically acceptable carrier; l) a kit for measuring the activityand/or the presence of a chemokine-like polypeptide comprising one ormore of the following reagents set forth in a) to k); m) a transgenicanimal cell that has been transformed with a vector or a nucleic acidaccording to d) or e), having constitutive or inducible alteredexpression levels of a polypeptide according to any one of a1) to a8);or n) a transgenic non-human animal that has been transformed to haveenhanced or reduced expression levels of a polypeptide according to a1)to a8).
 43. The composition of matter of claim 42, wherein the nucleicacid molecule in said vector is operatively linked to expression controlsequences allowing expression in prokaryotic or eukaryotic host cells ofthe encoded polypeptide.
 44. The composition of matter of claim 42,wherein said polypeptides or ligands are in the form of activeconjugates or complexes with a molecule chosen from radioactive labels,fluorescent labels, biotin, or cytotoxic agents.
 45. The composition ofmatter of claim 42, wherein the compound that reduces the expressionlevel of a polypeptide is an antisense oligonucleotide or a smallinterfering RNA.
 46. A process for producing cells that produce apolypeptide comprising transforming a cell with a polynucleotideselected encoding SEQ ID NO: 2, 4, 6, 8, 10, 12, 14, or 16 and culturingsaid cells to produce a polypeptide.
 47. A method of using a compositionof matter comprising obtaining a composition of matter according toclaim 42 and using said composition of matter in a method selected from:making a genetically engineered cell; producing a polypeptide; producinga pharmaceutical composition; the treatment of a disease; screeningcandidate compounds; identifying a candidate compound; or determiningthe activity, presence or both activity or presence of said compositionof matter.
 48. The method of claim 47, comprising geneticallyengineering cells with composition of matter comprising a vector or anucleic acid according to claim
 42. 49. The method of claim 48, furthercomprising culturing said cell under conditions in which the nucleicacid or vector is expressed, and recovering the polypeptide encoded bysaid nucleic acid or vector from the culture.
 50. The method of claim47, comprising the administration of a therapeutically effective amountof a composition of matter comprising a polypeptide, a ligand, anantagonist, a peptide mimetic, a nucleic acid, a cell, or a compound asset forth in claim 42 to an individual for the treatment of a disease.51. The method of claim 47, comprising a composition of mattercomprising a polypeptide, a ligand, an antagonist, a peptide mimetic, anucleic acid, a cell, or a compound as set forth in claim 42 with apharmaceutically acceptable carrier to produce a pharmaceuticalcomposition.
 52. The method of claim 47, wherein said method screens forcandidate compounds effective to treat a disease related to thechemokine-like polypeptides and comprises: a) contacting a cell, atransgenic animal cell, or a transgenic non-human animal as set forth inclaim 42 and having enhanced or reduced expression levels of thepolypeptide with a candidate compound; and b) determining the effect ofthe compound on the animal or on the cell.
 53. The method of claim 47,wherein said method identifies a candidate compound as anantagonist/inhibitor or agonist/activator of a polypeptide of claim 42and comprises: a) contacting said polypeptide of claim 42, saidcompound, and a mammalian cell or a mammalian cell membrane capable ofbinding the polypeptide; and b) measuring whether the molecule blocks orenhances the interaction of the polypeptide, or the response thatresults from such interaction, with the mammalian cell or the mammaliancell membrane.
 54. The method of claim 47, said method determining theactivity and/or the presence of a polypeptide in a sample, the methodcomprising: a) providing a protein-containing sample; b) contacting saidsample with a ligand of claim 1; and c) determining the presence of saidligand bound to said polypeptide.
 55. The method of claim 47, saidmethod determining the presence or the amount of a transcript or of anucleic acid encoding a polypeptide and comprising: a) providing anucleic acid-containing sample; b) contacting said sample with a nucleicacid of claim 42; and determining the hybridization of said nucleic acidwith a nucleic acid into the sample.
 56. The method of claim 55, wherinsaid contacting comprises the use of the primer sequences comprising SEQID NO: 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27 or 28 for determiningthe presence or the amount of a transcript or of a nucleic acid encodingthe polypeptide.
 57. The method of claim 56, wherein the amount of saidtranscript or nucleic acid is determined by Polymerase Chain Reaction.